Biologicals heretofore have been subjected to filtration for the purpose of sterilization. However, filtration does not always remove small bacteria, nor will bacteriological filters retain mycoplasma, or viruses. Fedoroff, S., Evans, V. J., Hopps, H. E., Sanford, K. K. and Boone, C. W. Summary of proceedings of a workshop on serum for tissue culture purposes. In Vitro: Vol. 7, 1972, p. 161-167. Barile, M. F. and Kern, J. Isolation of Mycoplasma arginini from commercial bovine sera and its implication in contaminated cell cultures. Proc. Soc. Exp. Biol. & Med. Vol. 138, 1971, p. 432-437.
For example, the sterilization of trypsin for use in digestion of mammalian tissues and tissue and cell cultures and for other purposes or the sterilization of other biologicals poses a serious problem. For instance, and as set forth in the foregoing publications, trypsin used to disperse secondary, continuous normal and continuous transformed tissue and cell cultures or other biologicals cannot be efficiently sterilized by conventional filtration through 0.22 micron filters. Further, as set forth in the foregoing publications, such bacteriological filtrates often contain adventitious viruses, mycoplasma and minute forms of pseudomonas and other bacteria which pass defective membrane pores. The presence of these contaminants poses a serious problem in all types of biological research and development requiring sterile or non-contaminated biological fluids.
In addition to the above problems, the repeated filtration of trypsin or other enzymes during the processes of clarification often allows the enzyme to autolyze, adding even more materially to the cost of the product, and the prolonged period of time required to clarify trypsin or other biologicals at ambient temperatures allows bacteria present therein to synthesize a variety of toxins which are then collected in the filtrate since they are smaller than the pores of filters used for sterilization. Even though bacterially sterile by filtration, biologicals that once contained bacteria may be spoiled for their original purposes because the product has now become cytotoxic and pyrogenic, and also may and often does contain agents which pass the filter, e.g., viruses and mycoplasma, as set forth in the foregoing publications.
Although acidic pH levels have been shown in the past to inactivate a variety of microorganisms, such application for biologics, i.e., human serum products, enzymes, hormones, antibiotics and other biologic fluids, etc., has never been successful or made feasible for sterilization of such fluids in the past. The parameters for complete sterilization of bacteria, viruses, spores, fungi and molds, yeast, mycoplasma, and other microbial flora previously have not been attained. In addition, the effects of acidic pH levels as ordinarily applied in the past fail to inactivate the very resistant aerobic and anaerobic spore-forming bacterium.
It would be highly advantageous to provide and the present invention is directed to a process of purifying and sterilizing biologicals without deleteriously affecting the biological, e.g., the proteolytic activity of the enzymes, by a relatively simple and inexpensive process in which the biological is acidified to a pH of not over about 1.0 and the acidified biological is stored at temperatures and for periods of time sufficient to inactivate the microbial flora without deleteriously affecting the biological or its proteolytic activity.